Our ultimate goal is to develop novel and effective treatments of glioblastoma, the most common and lethal human brain cancer. To achieve this goal, we propose to develop the SUMO1 inhibition compound (SMIC1) as a new anticancer drug for glioblastoma therapy. SUMO1 (small ubiquitin-like modifier-1) is a small regulatory protein that is linked to substrate proteins through enzymatic reactions. SUMO1 conjugation of its substrate proteins controls the cellular function of substrate proteins. In our recent work, we have revealed that SUMO1 conjugation pathway is overactive in glioblastoma and drives the cancer progression. To target this pathway, we have developed glioblastoma cell-based SUMO1 assays for drug screening and identified the SMIC1 from the NCI drugable compound library. In the efforts of preclinical development of SMIC1 as an anticancer drug, we have followed the FDA guidance for nonclinical evaluation of new anticancer agents and tested the toxicity and pharmacokinetics (PK) of SMIC1 and demonstrated that SMIC1 has an acceptable safety margin and drugable PK features in animals. In systemic administration, SMIC1 can be quickly delivered to brains through the blood brain barrier (BBB) and effectively inhibits glioblastoma xenograft growth. In search of target proteins, we have shown that cyclin-dependent kinase-6 (CDK6) is a substrate of both SUMO1 and ubiquitin (UB). SUMO1-CDK6 conjugation blocks CDK6 ubiquitination and the UB-mediated degradation and thus stabilizes CDK6 kinase for driving cell growth through phosphorylation of retinoblastoma protein-1 (RB1); thus, SMIC1 treatment blocks SUMO1-CDK6 conjugation and eliminates CDK6-RB1 pathway. On the other hand, CDK4/6 inhibitors have been developed targeting CDK4/6-RB1 pathway and they are now in clinic for cancer therapies. RB1 deletion and mutation occurs in about 11% glioblastomas and results in the cancer resistance to CDK4/6 inhibitors. In contrast, we have shown that SMIC1 can overcome the resistance through inhibition of various SUMO1 substrate proteins. The objective of this proposal is to develop SMIC1 as a new anticancer drug for treatment of glioblastomas. To achieve this, we will first determine the molecular mechanisms of action of SMIC1 in treatment of glioblastoma cells. In particular, we will examine how SMIC1 treatment induces the ubiquitination and degradation of SUMO1 protein and abolishes SUMO1 conjugation pathway in human glioblastoma cells. Next, we will examine the bioactivity and pharmacodynamics of SMIC1 in comparison with CDK4/6 inhibitors in genetically heterogenous glioblastoma cells and thus determine why SMIC1 treatment can overcome the resistance of RB1 deletion and mutation. Finally, we will evaluate the therapeutic efficacy of SMIC1 using the cancer stem cell cultures and xenograft models generated from patients' glioblastoma tissues. Upon completion, this project will lead to the genesis of a new class anticancer drug for clinical treatment of glioblastoma patients.